1. Field of the Invention
The invention relates to cellular controls for hemoglobin and, more specifically, to compositions and methods for generating suitable cellular, glycated hemoglobin A1c (Hb A1c) controls. In particular, cellular Hb A1c controls generated using a variety of methods are disclosed.
2. Description of Related Art
Hemoglobin (Hb) is a respiratory molecule found in red blood cells. It is responsible for transporting oxygen from the lungs to body cells and for transporting carbon dioxide from body cells to the lungs. Hemoglobin has a molecular weight of 64,000 Daltons and contains four polypeptide chains. Each chain binds to a heme group which consists of a tetrapyrrole ring chelated to an Fe2+ ion. In the lungs, the iron atoms of the hemoglobin molecule reversibly combine with an oxygen molecule, which is then transported to body cells as blood circulates. The oxygen is released from the hemoglobin molecule in the tissues, and then the oxygen-free hemoglobin molecule picks up carbon dioxide, which is transported back to the lungs, where it is released.
Hemoglobin is produced from cells in the bone marrow that become red blood cells. Certain illnesses result in a deficiency of hemoglobin, such as anemia and sickle cell disease. Still other diseases, such as polycythemia or erythrocytosis, result in excessive levels of hemoglobin. Therefore, as an aid in the diagnosis or monitoring of such diseases, methods and devices for determining the concentration of hemoglobin in whole blood are valuable.
Hemoglobin may be modified by the free glucose present in human plasma to form glycated hemoglobin (GHB). Hemoglobin A1c (Hb A1c, also referred to as A1c), constituting approximately 80% of all glycated Hb, is generated by the spontaneous reaction of glucose with the N-terminal amino group of the Hb A beta chain. The Hb A1c and the total glycated Hb values have a high degree of correlation, and either value may be used, for example in the management of treating diabetes. Formation of Hb A1c is slow but irreversible, and the blood level depends on both the life span of the red blood cells (average 120 days) and the blood glucose concentration. Therefore, Hb A1c represents the time-averaged blood glucose values over the preceding 2 to 3 months, and is not subject to wide fluctuations observed in blood glucose values. With respect to diabetes management, studies have shown that quality of life improves with decreasing levels of Hb A1c, and measurements every 3 to 6 months are recommended.
The determination of total hemoglobin is indicative of the oxygen-carrying capacity of whole blood. An ability to measure hemoglobin in blood samples is an essential part of diagnostic analysis and is also important for monitoring responsiveness to therapies directed towards diseases that affect hemoglobin and to therapies that are directed towards other diseases but which may have adverse side effects on the hemoglobin level.
The numerous methods and devices for the determination of hemoglobin include both direct analysis, i.e., analysis without prior modification of the hemoglobin, and indirect analysis. An example of a direct analysis method is the Tallquist Method, wherein a measurement of the transmission or reflection optical density of the red color imparted by oxyhemoglobin, the natural form of hemoglobin, is obtained. An example of an indirect analysis method is the Drabkin's Method. In this method, the iron in hemoglobin is oxidized with a ferricyanide to form methemoglobin, which is converted with a cyanide molecule to cyanomethemoglobin, which is then measured spectrophotometrically. It is important to accurately determine the total hemoglobin in the Hb A1c assay, because A1c is often reported as a fraction of the total hemoglobin.
Multiple Hb A1c assay methodologies have been developed since late 1970s. One of the standard methods for measuring Hb A1c uses ionic-exchange high performance liquid chromatography (HPLC), which separates and analyzes Hb A1c and other minor Hb components from unmodified hemoglobin (Hb A0) based upon their differences in chemical charges. A second methodology for detection of Hb A1c is designed by immunoinhibition turbidimetric techniques. The HbA1c assay in immunoassay includes an antibody-antigen reaction and a following turbidity measurement. The third methodology is boronate affinity chromatography, which utilizes a gel matrix containing immobilized boronic acid to capture the cis-diol group of glycated hemoglobin. The variety of Hb A1c testing methodologies requires a novel control that could be used in various methods and devices for detecting Hb A1c levels.
In most of the available methods, the first step for measuring Hb A1c levels is the manual or automatic production of a hemolysate by lysing the red blood cells with a special lytic reagent. Therefore, there is an ongoing need for cellular Hb A1c standards or controls that exhibit a similar matrix to that of patient specimens and that function in the analytical testing phases during an Hb A1c assay.
Currently, there are a number of Hb A1c normal and abnormal controls on the market. Almost all of these hemoglobin A1c controls are in the form of protein powders (lyophilized) or hemolyzed liquid solutions. Although these A1c controls have been in the market for a long time, they have shown various limitations: (1) none of these controls provide information about RBC lysis, one of the required and critical QC steps; (2) the stability of the lyophilized controls upon rehydration (after the first use) is as short as 1-2 weeks, although the protein powders can be stored for long periods of time at −20° C.; and (3) none of the currently manufactured hemolyzed liquid controls can be applied to ionic exchange HPLC methods, the main method of Hb A1c testing. Thus, there is a need for cellular (whole cell, or mimics whole cells) and stable Hb A1c controls that can be used with all testing methodologies.
The present invention relates to developing normal and abnormal high cellular Hb A1c controls that have the following advantages over previous controls: (1) they will work with at least the current Hb A1c detection methodologies and systems; (2) in certain embodiments they will have an Hb A1c value of about 10% or higher for the abnormal high (Level II) control; (3) in certain embodiments they will be substantially intact erythrocytes and have at least about 3 to 12 months of stability; and (4) they will mimic the whole blood sample. In contrast to the short stability of the protein solution exhibited by rehydrated lyophilized controls, the cellular Hb A1c controls of the present invention (also referred to as being cellular, whole cell, or in-cell) will have a much longer stability period (at least from about 3-12 months) and will be easy to use. On the other hand, in contrast to the hemolyzed nature and limited usage of hemolyzed liquid controls, the cellular Hb A1c controls, containing intact RBCs, will be able to provide a complete control for the foreseeable QC steps and will be utilized for currently known and available testing methodologies.